Prem Kumar Govindappa

Interests

Research

I am an academic veterinary research scientist. My area of specialization is in multidisciplinary translational and regenerative medicine, with a focus on studying the repurposing effect of the USFDA-approved 4-aminopyridine and Erythropoietin in trauma conditions of peripheral nerve, skeletal muscle, skin, bone, and intestines.

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Scholarly Contributions

Journals/Publications

• Govindappa, P. K., Begom, M., Gupta, Y., Elfar, J. C., Rawat, M., & Elfar, W. (2023). A critical role for erythropoietin on vagus nerve Schwann cells in intestinal motility. BMC biotechnology, 23(1), 12.
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  Dysmotility and postoperative ileus (POI) are frequent major clinical problems post-abdominal surgery. Erythropoietin (EPO) is a multifunctional tissue-protective cytokine that promotes recovery of the intestine in various injury models. While EPO receptors (EPOR) are present in vagal Schwann cells, the role of EPOR in POI recovery is unknown because of the lack of EPOR antagonists or Schwann-cell specific EPOR knockout animals. This study was designed to explore the effect of EPO via EPOR in vagal nerve Schwann cells in a mouse model of POI.
• Govindappa, P. K., Jagadeeshaprasad, M. G., Tortora, P., Talukder, M. A., & Elfar, J. C. (2023). Effects of 4-Aminopyridine on Combined Nerve and Muscle Injury and Bone Loss. The Journal of hand surgery, 48(8), 831.e1-831.e9.
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  Musculoskeletal injuries are common, and peripheral nerve injury (PNI) causes significant muscle and bone loss within weeks. After PNI, 4-aminopyridine (4-AP) improves functional recovery and muscle atrophy. However, it is unknown whether 4-AP has any effect on isolated traumatic muscle injury and PNI-induced bone loss.
• Lee, J. I., Talukder, M. A., Karuman, Z., Gurjar, A. A., Govindappa, P. K., Guddadarangaiah, J. M., Manto, K. M., Wandling, G. D., Hegarty, J. P., Waning, D. L., & Elfar, J. C. (2023). Functional recovery and muscle atrophy in pre-clinical models of peripheral nerve transection and gap-grafting in mice: effects of 4-aminopyridine. Neural regeneration research, 18(2), 439-444.
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  We recently demonstrated a repurposing beneficial effect of 4-aminopyridine (4-AP), a potassium channel blocker, on functional recovery and muscle atrophy after sciatic nerve crush injury in rodents. However, this effect of 4-AP is unknown in nerve transection, gap, and grafting models. To evaluate and compare the functional recovery, nerve morphology, and muscle atrophy, we used a novel stepwise nerve transection with gluing (STG), as well as 7-mm irreparable nerve gap (G-7/0) and 7-mm isografting in 5-mm gap (G-5/7) models in the absence and presence of 4-AP treatment. Following surgery, sciatic functional index was determined weekly to evaluate the direct in vivo global motor functional recovery. After 12 weeks, nerves were processed for whole-mount immunofluorescence imaging, and tibialis anterior muscles were harvested for wet weight and quantitative histomorphological analyses for muscle fiber cross-sectional area and minimal Feret's diameter. Average post-injury sciatic functional index values in STG and G-5/7 models were significantly greater than those in the G-7/0 model. 4-AP did not affect the sciatic functional index recovery in any model. Compared to STG, nerve imaging revealed more misdirected axons and distorted nerve architecture with isografting. While muscle weight, cross-sectional area, and minimal Feret's diameter were significantly smaller in G-7/0 model compared with STG and G-5/7, 4-AP treatment significantly increased right TA muscle mass, cross-sectional area, and minimal Feret's diameter in G-7/0 model. These findings demonstrate that functional recovery and muscle atrophy after peripheral nerve injury are directly related to the intervening nerve gap, and 4-AP exerts differential effects on functional recovery and muscle atrophy.
• Govindappa, P. K., & Elfar, J. C. (2022). Erythropoietin promotes M2 macrophage phagocytosis of Schwann cells in peripheral nerve injury. Cell death & disease, 13(3), 245.
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  Following acute sciatic nerve crush injury (SNCI), inflammation and the improper phagocytic clearance of dying Schwann cells (SCs) has effects on remodeling that lead to morbidity and incomplete functional recovery. Therapeutic strategies like the use of erythropoietin (EPO) for peripheral nerve trauma may serve to bring immune cell phagocytotic clearance under control to support debris clearance. We evaluated EPO's effect on SNCI and found EPO treatment increased myelination and sciatic functional index (SFI) and bolstered anti-apoptosis and phagocytosis of myelin debris via CD206 macrophages when compared to saline treatment. EPO enhanced M2 phenotype activity, both in bone marrow-derived macrophages (BMMØs) and peritoneal-derived macrophages (PMØs) in vitro, as well as in PMØs in vivo. EPO increased efferocytosis of apoptotic sciatic nerve derived Schwann cells (SNSCs) in both settings as demonstrated using immunofluorescence (IF) and flow cytometry. EPO treatment significantly attenuated pro-inflammatory genes (IL1β, iNOS, and CD68) and augmented anti-inflammatory genes (IL10 and CD163) and the cell-surface marker CD206. EPO also increased anti-apoptotic (Annexin V/7AAD) effects after lipopolysaccharide (LPS) induction in macrophages. Our data demonstrate EPO promotes the M2 phenotype macrophages to ameliorate apoptosis and efferocytosis of dying SCs and myelin debris and improves SN functional recovery following SNCI.
• Jagadeeshaprasad, M. G., Govindappa, P. K., Nelson, A. M., & Elfar, J. C. (2022). Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin. Journal of visualized experiments : JoVE.
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  This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.
• Jagadeeshaprasad, M. G., Govindappa, P. K., Nelson, A. M., Noble, M. D., & Elfar, J. C. (2022). 4-Aminopyridine Induces Nerve Growth Factor to Improve Skin Wound Healing and Tissue Regeneration. Biomedicines, 10(7).
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  The discovery of ways to enhance skin wound healing is of great importance due to the frequency of skin lesions. We discovered that 4-aminopyridine (4-AP), a potassium channel blocker approved by the FDA for improving walking ability in multiple sclerosis, greatly enhances skin wound healing. Benefits included faster wound closure, restoration of normal-appearing skin architecture, and reinnervation. Hair follicle neogenesis within the healed wounds was increased, both histologically and by analysis of K15 and K17 expression. 4-AP increased levels of vimentin (fibroblasts) and alpha-smooth muscle actin (α-SMA, collagen-producing myofibroblasts) in the healed dermis. 4-AP also increased neuronal regeneration with increased numbers of axons and S100 Schwann cells (SCs), and increased expression of SRY-Box Transcription Factor 10 (SOX10). Treatment also increased levels of transforming growth factor-β (TGF-β), substance P, and nerve growth factor (NGF), important promoters of wound healing. In vitro studies demonstrated that 4-AP induced nerve growth factor and enhanced proliferation and migration of human keratinocytes. Thus, 4-AP enhanced many of the key attributes of successful wound healing and offers a promising new approach to enhance skin wound healing and tissue regeneration.
• Lee, J. I., Govindappa, P. K., Wandling, G. D., & Elfar, J. C. (2022). Traumatic Peripheral Nerve Injury in Mice. Journal of visualized experiments : JoVE.
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  Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve and denervating muscle have long been a goal in musculoskeletal research. Many traumatically injured limbs have nerve trauma that defines the long-term patient outcome. Over several years, precise methods of producing microsurgical nerve injuries have been developed, including crush, lacerations, and nerve-gap grafting, allowing for reproducible outcome assessments. Moreover, newer methods are created for calibrated crush injuries that offer clinically relevant correlations with outcomes used to assess human patients. The principles of minimal manipulation to ensure low variability in nerve injury allow for adding still more associated tissue injuries into these models. This includes direct muscle crush and other components of limb injury. Finally, atrophy assessment and precise analysis of behavioral outcomes make these methods a complete package for studying musculoskeletal trauma that realistically incorporates all the elements of human traumatic limb injury.
• Lee, J. I., Wandling, G. D., Talukder, M. H., Govindappa, P. K., & Elfar, J. C. (2022). A Novel Standardized Peripheral Nerve Transection Method and a Novel Digital Pressure Sensor Device Construction for Peripheral Nerve Crush Injury. Bio-protocol, 12(5), e4350.
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  Peripheral nerve injury (PNI) is common in all walks of life, and the most common PNIs are nerve crush and nerve transection. While optimal functional recovery after crush injury occurs over weeks, functional recovery after nerve transection with microsurgical repair and grafting is poor, and associated with permanent disability. The gold-standard treatment for nerve transection injury is microsurgical tensionless end-to-end suture repair. Since it is unethical to do experimental PNI studies in humans, it is therefore indispensable to have a simple, reliable, and reproducible pre-clinical animal model for successful evaluation of the efficacy of a novel treatment strategy. The objective of this article is two-fold: (A) To present a novel standardized peripheral nerve transection method in mice, using fibrin glue for modeling peripheral nerve transection injury, with reproducible gap distance between the severed nerve ends, and (B) to document the step-wise description of constructing a pressure sensor device for crush injury pressure measurements. We have successfully established a novel nerve transection model in mice using fibrin glue, and demonstrated that this transection method decreases surgical difficulties and variability by avoiding microsurgical manipulations on the nerve, ensuring the reproducibility and reliability of this animal model. Although it is quite impossible to exactly mimic the pathophysiological changes seen in nerve transection with sutures, we hope that the close resemblance of our novel pre-clinical model with gold-standard suturing can be easily reproduced by any lab, and that the data generated by this method significantly contributes to better understanding of nerve pathophysiology, molecular mechanisms of nerve regeneration, and the development of novel strategies for optimal functional recovery. In case of peripheral nerve crush injury, current methods rely on inter-device and operator precision to limit the variation with applied pressure. While the inability to accurately quantify the crush pressure may result in reduced reproducibility between animals and studies, there is no documentation of a pressure monitoring device that can be readily used for real-time pressure measurements. To address this deficit, we constructed a novel portable device comprised of an Arduino UNO microcontroller board and force sensitive resistor (FSR) capable of reporting the real-time pressure applied to a nerve. This novel digital pressure sensor device is cheap, easy to construct and assemble, and we believe that this device will be useful for any lab performing nerve crush injury in rodents.
• Manto, K. M., Govindappa, P. K., Martinazzi, B., Han, A., Hegarty, J. P., Koroneos, Z., Talukder, M. A., & Elfar, J. C. (2022). Erythropoietin-PLGA-PEG as a local treatment to promote functional recovery and neurovascular regeneration after peripheral nerve injury. Journal of nanobiotechnology, 20(1), 461.
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  Traumatic peripheral nerve injury (TPNI) is a major medical problem with no universally accepted pharmacologic treatment. We hypothesized that encapsulation of pro-angiogenic erythropoietin (EPO) in amphiphilic PLGA-PEG block copolymers could serve as a local controlled-release drug delivery system to enhance neurovascular regeneration after nerve injury.
• Rodenhouse, A., Talukder, M. A., Lee, J. I., Govindappa, P. K., O'Brien, M., Manto, K. M., Lloyd, K., Wandling, G. D., Wright, J. R., Chen See, J. R., Anderson, S. L., Lamendella, R., Hegarty, J. P., & Elfar, J. C. (2022). Altered gut microbiota composition with antibiotic treatment impairs functional recovery after traumatic peripheral nerve crush injury in mice: effects of probiotics with butyrate producing bacteria. BMC research notes, 15(1), 80.
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  Antibiotics (ABX) are widely used for life-threatening infections and also for routine surgical operations. Compelling evidence suggests that ABX-induced alterations of gut microbiota composition, termed dysbiosis, are linked with diverse disease states including neurological and neurodegenerative conditions. To combat the consequences of dysbiosis, probiotics (PBX) are widely used. ABX-induced dysbiosis is reported to impair neurological function after spinal cord injury. Traumatic peripheral nerve injury (TPNI) results in profound neurologic impairment and permanent disability. It is unknown whether ABX treatment-induced dysbiosis has any impact on TPNI-induced functional recovery, and if so, what role medical-grade PBX could have on TPNI recovery.
• Lee, J. I., Gurjar, A. A., Talukder, M. A., Rodenhouse, A., Manto, K., O'Brien, M., Karuman, Z., Govindappa, P. K., & Elfar, J. C. (2021). Purposeful Misalignment of Severed Nerve Stumps in a Standardized Transection Model Reveals Persistent Functional Deficit With Aberrant Neurofilament Distribution. Military medicine, 186(Suppl 1), 696-703.
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  Functional recovery following primary nerve repair of a transected nerve is often poor even with advanced microsurgical techniques. Recently, we developed a novel sciatic nerve transection method where end-to-end apposition of the nerve endings with minimal gap was performed with fibrin glue. We demonstrated that transected nerve repair with gluing results in optimal functional recovery with improved axonal neurofilament distribution profile compared to the end-to-end micro-suture repair. However, the impact of axonal misdirection and misalignment of nerve fascicles remains largely unknown in nerve-injury recovery. We addressed this issue using a novel nerve repair model with gluing.
• Manto, K. M., Govindappa, P. K., Parisi, D., Karuman, Z., Martinazzi, B., Hegarty, J. P., Talukder, M. A., & Elfar, J. C. (2021). (4-Aminopyridine)-PLGA-PEG as a Novel Thermosensitive and Locally Injectable Treatment for Acute Peripheral Nerve Injury. ACS applied bio materials, 4(5), 4140-4151.
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  Traumatic peripheral nerve injury (TPNI) represents a major medical problem that results in loss of motor and sensory function, and in severe cases, limb paralysis and amputation. To date, there are no effective treatments beyond surgery in selective cases. In repurposing studies, we found that daily systemic administration of the FDA-approved drug 4-aminopyridine (4-AP) enhanced functional recovery after acute peripheral nerve injury. This study was aimed at constructing a novel local delivery system of 4-AP using thermogelling polymers. We optimized a thermosensitive (4-AP)-poly(lactide--glycolide)--poly(ethylene glycol)--poly(lactide--glycolide) (PLGA-PEG-PLGA) block copolymer formulation. (4-AP)-PLGA-PEG exhibited controlled release of 4-AP both and for approximately 3 weeks, with clinically relevant safe serum levels in animals. Rheological investigation showed that (4-AP)-PLGA-PEG underwent a solution to gel transition at 32 °C, a physiologically relevant temperature, allowing us to administer it to an injured limb while subsequently forming an gel. A single local administration of (4-AP)-PLGA-PEG remarkably enhanced motor and sensory functional recovery on post-sciatic nerve crush injury days 1, 3, 7, 14, and 21. Moreover, immunohistochemical studies of injured nerves treated with (4-AP)-PLGA-PEG demonstrated an increased expression of neurofilament heavy chain (NF-H) and myelin protein zero (MPZ) proteins, two major markers of nerve regeneration. These findings demonstrate that (4-AP)-PLGA-PEG may be a promising long-acting local therapeutic agent in TPNI, for which no pharmacologic treatment exists.
• Patil, M., Saheera, S., Dubey, P. K., Kahn-Krell, A., Kumar Govindappa, P., Singh, S., Tousif, S., Zhang, Q., Lal, H., Zhang, J., Qin, G., & Krishnamurthy, P. (2021). Novel Mechanisms of Exosome-Mediated Phagocytosis of Dead Cells in Injured Heart. Circulation research, 129(11), 1006-1020.
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  [Figure: see text].
• Talukder, M. A., Lee, J. I., Hegarty, J. P., Gurjar, A. A., O'Brien, M., Karuman, Z., Wandling, G. D., Govindappa, P. K., & Elfar, J. C. (2021). Obligatory role of Schwann cell-specific erythropoietin receptors in erythropoietin-induced functional recovery and neurogenic muscle atrophy after nerve injury. Muscle & nerve, 63(2), 268-272.
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  Erythropoietin (EPO) promotes myelination and functional recovery in rodent peripheral nerve injury (PNI). While EPO receptors (EpoR) are present in Schwann cells, the role of EpoR in PNI recovery is unknown because of the lack of EpoR antagonists or Schwann cell-specific EpoR knockout animals.
• Wandling, G. D., Lee, J. I., Talukder, M. A., Govindappa, P. K., & Elfar, J. C. (2021). Novel Real-time Digital Pressure Sensor Reveals Wide Variations in Current Nerve Crush Injury Models. Military medicine, 186(Suppl 1), 473-478.
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  Peripheral nerve crush injury (PNCI) models are commonly used to study nerve damage and the potential beneficial effects of novel therapeutic strategies. Current models of PNCI rely on inter-device and operator precision to limit the variation with applied pressure. Although the inability to accurately quantify the PNCI pressure may result in reduced reproducibility between animals and studies, there is very limited information on the standardization and quantification of applied pressure with PNCI. To address this deficit, we constructed a novel device comprised of an Arduino UNO microcontroller board and Force Sensitive Resistor capable of reporting the real-time pressure applied to a nerve.
• Govindappa, P. K., Joladarashi, D., Hallur, R. L., Sanganal, J. S., & Phani, A. R. (2020). Toxicity evaluation of 6-mercaptopurine-Chitosan nanoparticles in rats. Saudi pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society, 28(1), 147-154.
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  The 6-mercaptopurine (6-MP) is an effective immunosuppressant and anti-cancer drug. However, the usage of 6-MP is limited due to its well-known side effects, such as myelotoxicity and hepato-renal toxicity. To curtail the potential toxic effects, we have used chitosan as a natural biodegradable and biocompatible polysaccharide to synthesize 6-Mercaptopurine-Chitosan Nanoparticles (6-MP-CNPs).
• Govindappa, P. K., Patil, M., Garikipati, V. N., Verma, S. K., Saheera, S., Narasimhan, G., Zhu, W., Kishore, R., Zhang, J., & Krishnamurthy, P. (2020). Targeting exosome-associated human antigen R attenuates fibrosis and inflammation in diabetic heart. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(2), 2238-2251.
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  RNA-binding proteins like human antigen R (HuR) are key regulators in post-transcriptional control of gene expression in several pathophysiological conditions. Diabetes adversely affects monocyte/macrophage biology and function. It is not known whether diabetic milieu affects cellular/exosome-HuR and its implications on cardiac inflammation and fibrosis. Here, we evaluate in vitro and in vivo effects of diabetic milieu on macrophage cellular/exosome-HuR, alterations in intercellular cross talk with fibroblasts, and its impact on cardiac remodeling. Human failing hearts show higher HuR levels. Diabetic milieu activates HuR expression in cardiac- and cultured bone marrow-derived macrophages (BMMØ) and stimulates HuR nuclear-to-cytoplasmic translocation and exosome transfer. Exosomes from macrophages exposed to diabetic milieu (high glucose or db/db mice) significantly increase inflammatory and profibrogenic responses in fibroblast (in vitro) and cardiac fibrosis in mice. Intriguingly, Exo-HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with BMMØ-derived HuR-deficient exosomes inhibits angiotensin II-induced cardiac fibrosis response and preserves left ventricle function as compared to control-exosome administration. To the best of our knowledge, this is the first study to demonstrate that diabetes activates BMMØ HuR expression and its transfer into exosome. The data suggest that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes.
• Govindappa, P. K., Talukder, M. A., Gurjar, A. A., Hegarty, J. P., & Elfar, J. C. (2020). An effective erythropoietin dose regimen protects against severe nerve injury-induced pathophysiological changes with improved neural gene expression and enhances functional recovery. International immunopharmacology, 82, 106330.
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  The functional recovery following non-severing peripheral nerve injury (PNI) is often incomplete. Erythropoietin (EPO) is a pleiotropic hormone and it has been shown to protect peripheral nerves following mild and even moderate severity injuries. However, the effectiveness of EPO in severe PNI is largely unknown. In this study, we sought to investigate the neuroprotective effect of a new dose regimen of EPO in severe sciatic nerve crush injury (SSCI). Adult male mice (8 animals/group) were randomly assigned to sham (normal saline, 0.1 ml/mouse), SSCI (normal saline, 0.1 ml/mouse) and SSCI with EPO (5000 IU/kg) groups. SSCI was performed using calibrated forceps for 30 sec. EPO or normal saline was administered intraperitoneally immediately after the SSCI and at post-injury day1 and 2. The functional recovery after injury was assessed by sciatic function index (SFI), von Frey Test (VFT), and grip strength test. Mice were euthanized on day 7 and 21 and nerves at injury/peri-injury site were processed for gene (quantitative real-time PCR) and protein (immunohistochemistry) expression analysis. EPO significantly improved SFI, VFT, and hind limb paw grip strength from post-injury day 7. EPO demonstrated significant regulatory effects on mRNA expression of inflammatory (IL-1β and TNF-α), anti-inflammatory (IL-10), angiogenesis (VEGF and eNOS), and myelination (MBP) genes. The protein expression of IL-1β, F4/80, CD31, NF-κB p65, NF-H, MPZ, and DHE (redox-sensitive probe) was also significantly modulated by EPO treatment. In conclusion, the new dose regimen of EPO augments sciatic nerve functional recovery by mitigating inflammatory, anti-inflammatory, oxidative stress, angiogenesis, and myelination components of SSCI.
• Lee, J. I., Gurjar, A. A., Talukder, M. A., Rodenhouse, A., Manto, K., O'Brien, M., Govindappa, P. K., & Elfar, J. C. (2020). A novel nerve transection and repair method in mice: histomorphometric analysis of nerves, blood vessels, and muscles with functional recovery. Scientific reports, 10(1), 21637.
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  Peripheral nerve transection is associated with permanent functional deficit even after advanced microsurgical repair. While it is difficult to investigate the reasons of poor functional outcomes of microsurgical repairs in humans, we developed a novel pre-clinical nerve transection method that allows reliable evaluation of nerve regeneration, neural angiogenesis, muscle atrophy, and functional recovery. Adult male C57BL/6 mice were randomly assigned to four different types of sciatic nerve transection: Simple Transection (ST), Simple Transection & Glue (TG), Stepwise Transection and Sutures (SU), and Stepwise Transection and Glue (STG). Mice were followed for 28 days for sciatic function index (SFI), and sciatic nerves and hind limb muscles were harvested for histomorphological and cellular analyses. Immunohistochemistry revealed more directional nerve fiber growth in SU and STG groups compared with ST and TG groups. Compared to ST and TG groups, optimal neural vessel density and branching index in SU and STG groups were associated with significantly decreased muscle atrophy, increased myofiber diameter, and improved SFI. In conclusion, our novel STG method represents an easily reproducible and reliable model with close resemblance to the pathophysiological characteristics of SU model, and this can be easily reproduced by any lab.
• Rajashekaraiah, R., Kumar, P. R., Prakash, N., Rao, G. S., Devi, V. R., Metta, M., Narayanaswamy, H. D., Swamy, M. N., Satyanarayan, K., Rao, S., Rathnamma, D., Sahadev, A., Sunilchandra, U., Santhosh, C. R., Dhanalakshmi, H., Kumar, S. N., Ruban, S. W., Kalmath, G. P., Gomes, A. R., , Kumar, K. R., et al. (2020). Anticancer efficacy of 6-thioguanine loaded chitosan nanoparticles with or without curcumin. International journal of biological macromolecules, 148, 704-714.
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  6-Thioguanine encapsulated chitosan nanoparticles (6-TG-CNPs) has formulated by the ionic-gelation method. Morphologically, the 6-TG-CNPs were spherical and showed mean size, PDI, zeta potential, and entrapment efficiency of 261.63 ± 6.01 nm, 0.34 ± 0.10, +15.97 ± 0.46 mV and 44.27%, respectively. The IR spectra confirmed the 6-TG complex with chitosan. The in vitro drug release profile of 6-TG-CNPs revealed an increase in sustained-release (91.40 ± 1.08% at 48 h) at pH 4.8 compared to less sustained-release (73.96 ± 1.12% at 48 h) at pH 7.4. The MTT assay was conducted on MCF-7 and PA-1 cell lines at 48 h incubation to determine % cell viability. The IC values of 6-TG, 6-TG-CNPs, and curcumin for MCF-7 were 23.09, 17.82, and 15.73 μM, respectively. Likewise, IC values of 6-TG, 6-TG-CNPs, and curcumin for PA-1 were 5.81, 3.92, and 12.89 μM, respectively. A combination of 6-TG-CNPs (IC) with curcumin (IC) on PA-1 and MCF-7 showed % cell viability of 43.67 ± 0.02 and 49.77 ± 0.05, respectively. The in vitro cytotoxicity potential in terms of % cell viability, early apoptosis, G2/M phase arrest, and DNA demethylating activity of 6-TG-CNPs alone and combination with curcumin proved to be more effective than that of 6-TG on PA-1 cells.
• Govindappa, P. K. (2019). Effect of Withania somnifera on gentamicin induced renal lesions in rats. Brazilian Journal of Pharmacognosy. doi:https://doi.org/10.1016/j.bjp.2018.12.005
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  Gentamicin induced renal complications are well known in humans and animals. Medicinal properties of Withania somnifera (L.) Dunal, Solanaceae, are recognized to improve renal functions. However, the pharmacological function of W. somnifera is not completely understood. We sought to unravel medicinal therapeutic function of W. somnifera on gentamicin-induced nephrotoxicity in wistar rats. Twenty-four adult male wistar rats evenly divided into four groups to evaluate in vivo nephroprotective and nephrocurative function of W. somnifera in gentamicin induced nephrotoxic rats. Experimental design as follows: Group I, saline control for 21 days; Group II, gentamicin nephrotoxic control for eight days; Group III, alcoholic extract of W. somnifera for 13 days + simultaneous administration of gentamicin and W. somnifera, from day 14 to 21 (nephroprotective) and Group IV, gentamicin for 8 days + alcoholic extract of W. somnifera from day 9 to 21 (nephrocurative). End of experiment, respective serum and kidney tissue samples used to analyze renal function. Withania somnifera as a nephroprotective and nephrocurative molecule significantly restore the renal function on gentamicin-induced nephrotoxicity. This phenomenon is accompanied with significantly reduced blood urea nitrogen, creatine, alkaline phosphatase, gamma-glutamyl transferase, albumin, total protein, calcium, potassium and kidney malon-dialdehyde concentrations. Additionally, W. somnifera significantly increased antioxidant activities of glutathione and superoxide dismutase to protect renal tissue damage from gentamicin in wistar rats. Over all, W. somnifera treated nephroprotective animal shows improved recovery compared to nephrocuartive. The nephroprotective or nephrocurative effect of W. somnifera could be due to inherent antioxidant and free-radical-scavenging principle(s). In the near future, biologically active compounds of W. somnifera (withanolides) could appear as a novel therapeutic molecule for renal disorders.
• Govindappa, P. K. (2015). Anti-cancerous efficacy and pharmacokinetics of 6-mercaptopurine loaded chitosan nanoparticles. Pharmacological Research. doi:https://doi.org/10.1016/j.phrs.2015.07.025
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  6-Mercaptopurine is a cytotoxic and DLS, SEM, XRD and apoptosis assay, cell cycle arrest and ROS indices) on HT-1080 and MCF-7 cells. In vivo 
• Govindappa, P. K. (2015). Fabrication, Characterization and In Vitro Osteogenic Potential of Polyvinyl Pyrrolidone-Titania (PVP-TiO ) Nanofibers. Analytical Chemistry Letters. doi:https://doi.org/10.1080/22297928.2015.1048728
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  or bone tissue engineering applications Titanium is the material of choice owing to its biocompatible and osteoconductive nature. Biocompatibility TiO2 is due to its unique properties of attracting surface proteins of cell surface. Our present study was designed to evaluate optimum conditions to get homogenous nanofibers of titanium with minimum fiber diameter. All the formulations were subjected to SEM analysis. By using protocol 1 the minimum size of nanofibers we could get is around 320 nm (NF1C) and for protocol 2 we could get around 90 nm (NF2C). XRD analysis of formulations NF1C and NF2C proved that the titanium is in anatase phase. We also evaluated biocompatibility, cell proliferation and osteogenic potential of two formulations (NF1C and NF2C) using mouse preosteoblasts (MC3T3-E1). Osteogenic potential of NF1C and NF2C was evaluated by quantifying osteogenic markers of differentiation like ALP and calcium content. ALP activity was highly expressed in cells grown on NF1C and NF2C formulations but higher degree of expression was seen on NF2C. A similar trend was followed for calcium deposition when cells were cultured on NF1C and NF2C formulations for 7 days. Our experimental results indicated that nanofibrous TiO2 substrates not only supported cellular growth, adhesion, and proliferation but also can guide preosteoblasts to osteoblast lineage. These results may be useful in preparing osteoconductive scaffolding materials for bone tissue engineering applications.
• Govindappa, P. K. (2015). Wound healing potential of green synthesized silver nanoparticles prepared from Lansium domesticum fruit peel extract. Materials Express. doi:https://doi.org/10.1166/mex.2015.1225
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  Silver nanoparticles (AgNPs) are proved to be potential wound healing materials for normal and burn related wounds due to their antibacterial and antifungal properties. Although, the wound healing properties of AgNPs through green synthesis were less reported. In the present study, green synthesized AgNPs were incorporated in Pluronic F127 gels as a delivery system to evaluate their wound healing potentiality. Interestingly, the wound healing activity of 0.1% w/w (AgNPs) in Pluronic F127 gels was enhanced to a considerable extent which could be corroborated by increased amount of hydroxylproline content (2.39 ± 0.28 μg/mL), wound tensile strength (33.41 ± 2.38 N/cm2, and wound closure time, which were quite higher than other treated groups. Histopathology and biochemical tests did not show any inflammation, however, the amount of collagen production was increased in the group treated by 0.1% w/w AgNPs in Pluronic F127 gels.
• Govindappa, P. K. (2014). Polyvinylpyrrolidone oral films of enrofloxacin: film characterization and drug release. International Journal of Pharmaceutics. doi:10.1016/j.ijpharm.2014.05.033
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  Enrofloxacin is a fluoroquinolone derivative used for treating urinary tract, respiratory and skin infections in animals. However, low solubility and low bioavailability prevented it from using on humans. Polyvinylpyrrolidone (PVP) is an inert, non toxic polymer with excellent hydrophilic properties, besides it can enhance bioavailability by forming drug polymer conjugates. With the aim of increasing solubility and bioavailability, enrofloxacin thin films were prepared using PVP as a polymer matrix. The obtained oral thin films exhibited excellent uniformity and mechanical properties. Swelling properties of the oral thin films revealed that the water uptake was enhanced by 21%. The surface pH has been found to be 6.8±0.1 indicating that these films will not cause any irritation to oral mucosa. FTIR data of the oral thin films indicated physical interaction between drug and polymer. SEM analysis revealed uniform distribution of drug in polymer matrix. In vitro drug release profiles showed enhanced release profiles (which are also pH dependant) for thin films compared to pure drug. Antibacterial activity was found to be dose dependent and maximum susceptibility was found on Klebsiella pneumonia making this preparation more suitable for respiratory infections.
• Govindappa, P. K. (2014). Preparation and Characterization of Nanofibrous Solid Dosage Form Containing Enrofloxacin. Analytical Chemistry Letters. doi:https://doi.org/10.1080/22297928.2014.1000964
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  For the first time, we have prepared electrospun polyvinyl pyrrolidine (PVP) nanofibers loaded with enrofloxacin (PVP-ENRNF) using electrospinning technique. The morphology and physico-chemical properties were characterized by employing Scanning Electron Microscopy (SEM) and Fourier Transform InfraRed (FTIR) spectroscopy, respectively. Morphology by SEM revealed fiber diameter ranging from 450-725 nm. FTIR spectroscopy indicated physical interaction between the polymer and drug. Release profiles of enrofloxacin (ENR) from PVP-ENRNFs were studied, which controlled/influenced by the pH of the release media. Cumulative drug release profiles of PVP-ENRNFs at pH 3.8 (71.96 %), 6.4 (62.34 %) and 7.4 (54.14 %) were comparatively better than release profiles of pure ENR. These nanofibers exhibited potent antibacterial activity on tested microbes. These PVP-ENRNFs can be efficiently used as potential materials for oral as well as local delivery of enrofloxacin for treating respiratory and wound infections.

Presentations

• Govindappa, P. K. (2023, August). Erythropoietin is a Post-Traumatic Anti-Inflammatory Treatment that Supports Macrophage Recruitment, Transition, and Phagocytosis of Dead Cells Following Peripheral Nerve Injury in Mice.. Military Health System Research Symposium (MHSRS).
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  Introduction: Traumatic peripheral nerve injury (TPNI) is significant and occurs in approximately 3% of all trauma patients. Inflammation, apoptosis, and obliteration of myelin and axons are the primary pathophysiological consequences of TPNI. Infiltrating immune cells play a critical role following TPNI, and inflammation itself undergoes a transition from an initial pro-inflammatory reaction to an anti-inflammatory phase. This is critical for nerve repair and structural modification. However, a persistent and severe proinflammatory reaction can impair peripheral nerve recovery, which depends on macrophage infiltration and early phenotypic transitions for the phagocytosis of cellular debris in advance of Schwann-cell orchestration of nerve regeneration. There is an unmet clinical need for a therapeutic agent which can enhance functional recovery by controlling this inflammation after TPNI. Erythropoietin (EPO), a pleiotropic hormone approved by the U.S. Food and Drug Administration (FDA) for anemia treatment, seems to have a role in guiding this transition. EPO can mitigate inflammation and may exert control of macrophage inflammation and phagocytosis. The significance of the EPO treatment on cellular debris clearance in the nerve after the injury is unknown, despite our work and the work of others motivating clinical translation of EPO for TPNI. We hypothesized that EPO may affect the critical clearance activity of M2 phenotype macrophages after TPNI, which might explain EPO’s neuroregenerative potential. Materials and Methods: All animal experiments described conform to Institutional Animal Care and Use Committee (IACUC) approved protocols at The Pennsylvania State University College of Medicine, Hershey, PA. Ten-week-old male C57BL/6 J mice weighing 25 ± 3 g were anesthetized using intraperitoneal ketamine (100 mg/kg)/xylazine (10 mg/kg) anesthesia, under a stereo microscope, the sciatic nerve was exposed, and calibrated crush injury was performed ~3 mm proximal to the sciatic nerve trifurcation using jig-modified forceps (5 mm tip width) for 30 s. The skin was closed with surgical staples, and post-operative slow-release buprenorphine (0.05 mg/kg) was given subcutaneously to all animals as an analgesic. The experimental animals (n = 7 animals/group) were randomly assigned to Sham (normal saline, 0.1 ml/mouse), sciatic nerve crush injury (SNCI) (normal saline, 0.1 ml/mouse), and SNCI with EPO (5000 IU/kg; Epoetin alfa) groups. EPO was given intraperitoneally immediately after surgery and post-surgery days 1 and 2. Functional recovery following nerve injury was assessed using walking track analysis (WTA) on days 3 and 7. Mice were euthanized on post-injury days 3 and 7, and sciatic nerve was harvested from the ipsilateral hindlimbs for apoptosis, phagocytosis, and myelination analysis using immunofluorescence (IF) staining. In-vitro and ex-vivo phagocytosis of dead-Schwann cells was conducted using mouse bone marrow- and peritoneal-derived macrophages by IF and flow cytometry. The effect of EPO on the polarization of macrophages (M1 to M2 phenotypes) under lipopolysaccharide (LPS)-induced stress conditions were analyzed using qRT-PCR and flow cytometry. Data were analyzed using either one-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test or unpaired t-tests by GraphPad Prism Version 8.4.3. Results: EPO treatment significantly augmented anti-apoptosis and phagocytosis of myelin debris via CD206+ macrophages and increased myelination and sciatic functional index (SFI) compared to saline treatment after TPNI. EPO treatment showed a significant improvement in efferocytosis of apoptotic sciatic nerve-derived Schwann cells when cocultured with both bone marrow- and peritoneal-derived macrophages in-vitro. EPO also increased phagocytosis of dead Schwann cells in-vivo by peritoneal macrophages – cells totally outside the site of injury. EPO treatment significantly attenuated bone marrow-derived macrophage pro-inflammatory genes (IL1β, iNOS, and CD68) and augmented anti-inflammatory genes (IL10 and CD163) in addition to the cell surface marker CD206 expression after LPS induction. EPO also showed antiapoptotic (Annexin V/ 7AAD) effects under LPS conditions in bone marrow-derived macrophages. Our data demonstrate that EPO promotes the M2 phenotype macrophages to ameliorate sciatic nerve apoptosis and efferocytosis of dying Schwann cells and myelin debris and improves sciatic nerve functional recovery following SNCI. Conclusions: To our knowledge, this is the first study to demonstrate EPO’s ameliorative effect on the advancement of nerve regeneration and functional recovery following TPNI via M2 phenotype macrophage phagocytosis of Schwann cell debris with early anti-apoptotic and anti-inflammatory effects. These data may translate to EPO’s utility in the early phase post-injury to prevent destructive post-traumatic inflammation and promote recovery in the traumatized limb.
• Govindappa, P. K. (2023, August). Vagus nerve Schwann cell erythropoietin receptors are critical for early functional recovery of intestinal motility after postoperative ileus. American Neurogastroenterology and Motility Society (ANMS). Austin.
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  Background: Intestinal motility disorders (IMDs) are poorly understood and medically complex problems affecting an increasing number of patients. Postoperative ileus (POI) is a form of IMDs with frequent occurrence after abdominal surgery. Erythropoietin (EPO) is a multifunctional tissue-protective cytokine that promotes recovery of the intestine in various injuries. While EPO receptors (EPOR) are present on vagal Schwann cells, the role of EPOR in POI recovery is unknown because of the lack of EPOR antagonists or Schwann-cell specific EPOR knockout animals. This study was designed to explore the effect of EPO via EPOR on vagus nerve Schwann cells in a mouse model of POI.Methods: We used the Cre-loxP system to develop a myelin protein zero (Mpz) promoter-driven knockout mouse model of vagus nerve-Schwann cell EPOR (MpzCre-EPORflox/flox / Mpz-EPOR-KO confirmed using genotyping PCR and qRT-PCR. We then measured the intestinal transit time (ITT) at baseline and after induction of POI with and without EPO treatment.Results: Although we have previously shown that EPO accelerates functional recovery in POI in wild-type mice, EPO treatment did not improve functional recovery of ITT in POI of Mpz-EPOR-KO mice.Conclusions: This is the first pre-clinical study to demonstrate a novel mouse model of EPOR specific knock out on Schwan cells with an effect in the gut. We also showed novel beneficial effects of EPO through vagus nerve Schwann cell-EPOR in intestinal dysmotility. Our findings suggest that EPO-EPOR signaling in the vagus nerve after POI is important for the functional recovery of ITT.Keywords: Erythropoietin, Erythropoietin receptors, Vagus nerve, Schwann cells, Enteric glial cells, Intestinal transit time, Postoperative ileus
• Govindappa, P. K. (2022, May). Erythropoietin M2 macrophage Signaling Promotes Schwann Cells Clearance and Functional Recovery Following Peripheral Nerve Injury. Experimental Biology.
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  BackgroundTraumatic peripheral nerve injury (TPNI) obliterates axons and Schwann cells (SCs) that must be cleared through phagocytosis to allow surviving SCs to de-differentiate and divide and guide the regeneration process. In TPNI, macrophage (MØ) infiltration and early phenotypic transitions (M1 to M2) are critical for the phagocytosis of SCs debris. Delayed phagocytosis critically aggravates inflammation and impairs MØ function, which leads to failure to advance SC orchestration and neuronal regeneration. Erythropoietin (EPO), a pluripotent hormone, helps to guide MØs and SC transition post-TPNI. The significance of the effects of EPO on debris clearance, apoptosis, myelination, and functional improvement is unknown, despite much effort motivating clinical translation of EPO for TPNI. We hypothesized that a role in supporting M2 phenotype macrophages after TPNI might explain EPO’s neuroprotective function through SCs debris clearance.Methods and ResultsWe evaluated EPO’s effect on sciatic nerve crush injury (SNCI) and found EPO treatment (5000 IU/kg, intraperitoneally, post-surgery days 1, 2, and 3,) increased myelination and sciatic functional index (SFI) and augmented anti-apoptosis and phagocytosis of myelin debris via CD206+ macrophages when compared to saline treatment in the mouse. EPO increased efferocytosis of apoptotic sciatic nerve derived Schwann cells (SNSCs) both in bone marrow derived macrophages (BMMØs) and peritoneal derived macrophages (PMØs) in-vitro, as well as in PMØs in-vivo by immunofluorescence (IF) and flow cytometry. EPO treatment significantly attenuated BMMØ pro-inflammatory genes (IL1b, iNOS, and CD68) and augmented anti-inflammatory genes (IL10, and CD163) in addition to the cell surface marker CD206 expression after lipopolysaccharide (LPS) induction. EPO also showed anti-apoptotic (Annexin V/ 7AAD) effects in BMMØs. Our data demonstrate that EPO promotes the M2 phenotype macrophages to ameliorate SN apoptosis and efferocytosis of dying SCs and myelin debris and improves SN functional recovery following SNCI.ConclusionsTo the best of our knowledge, this is the first study to demonstrate EPO’s ameliorative effect on the progression of post-TPNI via M2 phenotype macrophage phagocytosis of SCs debris with early anti-apoptotic and anti-inflammatory effects. These data may support the findings of other researchers and might shed light on the role of EPO in improving functional recovery in the traumatized limb.
• Govindappa, P. K. (2021, May). Differential effects of 4-aminopyridine on nerve injury severity-dependent functional recovery and muscle atrophy in mice. Experimental Biology.
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  Peripheral nerve injury (PNI) represents a major public health problem that often leads to profound neurologic impairment, disuse muscle atrophy, and permanent disability. While the functional recovery following nerve transection and repair is disappointing, the impact of irreparable nerve gap remains poorly characterized. Recently we demonstrated a repurposing beneficial effect of 4-aminopyridine (4-AP), a potassium channel blocker, on the functional recovery and muscle atrophy after sciatic nerve crush injury in rodents. However, this effect of 4-AP is unknown in nerve transection and gap models. This study was designed to evaluate and compare the functional recovery and muscle atrophy between nerve transection and nerve gap models in the absence (saline) and presence of 4-AP treatment. Under deep anesthesia and aseptic conditions, complete nerve transection or nerve gap injury was performed in male C57BL/6 mice (n = 10/group). In novel Stepwise Nerve Transection and Glue (STG) group, right sciatic nerve was sequentially transected with fibrin gluing to prevent gap formation. In nerve gap model, a large gap was created by dissecting out 7 mm (Gap-7) of right sciatic nerve section where the proximal stump of the nerve was buried underneath the muscle with fibrin glue. Following surgery, 4-AP (40 µg, ip) or saline was given daily and sciatic function index (SFI) was determined weekly for 12 weeks to evaluate functional recovery. After 12 weeks, tibialis anterior (TA) muscles were harvested for wet weight and histomorphological analysis. To asses muscle atrophy, right TA (RTA) muscle mass is presented as a percentage of contralateral healthy left TA (LTA) muscle which was assigned 100%. We found that SFI in STG group significantly dropped from baseline -9.9±2.83 to -66±2.24 (P
• Govindappa, P. K. (2021, May). Local (4-Aminopyridine)-PLGA-PEG Treatment Improves Functional Recovery and Muscle Morphology after Traumatic Peripheral Nerve Injury in Mice. Experimental Biology.
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  Traumatic peripheral nerve injury (TPNI) represents a major medical problem resulting in loss of both motor and sensory function, and in severe cases, permanent disability. Inadequate axonal regeneration and delayed muscle reinnervation contribute to muscle atrophy and poor functional recovery. To date, there is no effective pharmacologic treatment which can promote neuromuscular outcomes after TPNI. In repurposing studies, we have shown that daily systemic 4-aminopyridine (4-AP), an FDA-approved potassium channel blocker for multiple sclerosis, enhances functional recovery, improves myelination, and attenuates muscle atrophy after TPNI. However, 4-AP at higher doses cause adverse effects such as seizures. Thus, we sought to develop a novel locally injectable formulation of 4-AP which could be administered to a nerve injury site and deliver 4-AP at a sustained rate for several weeks. We developed (4-AP)-Poly(lactide-co-glycolide)-b-Poly(ethylene glycol)-b-Poly(lactide-co-glycolide) (PLGA-PEG-PLGA), consisting of two FDA-approved, biodegradable, and biocompatible hydrogels. It is unknown whether this locally injectable formulation of 4-AP would have an impact on TPNI-induced functional recovery, as well as on muscle atrophy. In this study, an established mouse model of right sciatic nerve crush injury was used and mice received one of 4 treatments: (4-AP)-PLGA-PEG, PLGA-PEG vehicle, systemic 4-AP, or saline control (n = 5/group). Functional recovery was assessed using sciatic function index, grip strength, and von Frey filament testing. At post-injury day 28, gastrocnemius (GN) and tibialis anterior (TA) muscles were harvested from both injured (innervated by crushed nerve) and uninjured (contralateral healthy) hind limbs for histomorphological analysis. Our results show that a single local administration of (4-AP)-PLGA-PEG remarkably enhanced motor and sensory functional recovery on post-sciatic nerve crush injury days 1, 3, 7, 14, and 21. Histomorphometric analysis of the muscles revealed that (4-AP)-PLGA-PEG treatment significantly increased right TA cross-sectional area (CSA) (1488.2 µm2 vs. 438.4 µm2, p
• Govindappa, P. K. (2021, May). The Potential Repurposing Effect 4-Aminopyridine in Nerve and Muscle Injury-induced Bone Loss. Experimental Biology.
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  Muscle injuries are one of the most common injuries occurring in physical and sporting activities, and account for 10-55% of all injuries. Peripheral nerve injury (PNI) itself accounts for 3-5% of all injuries in trauma patients, and muscle injuries can be concurrent with PNI because of their close proximity. Importantly, the peripheral nervous system is critically involved in bone metabolism, bone mineralization, and bone remodeling, and PNI results in substantial bone loss. While first aid for muscle injuries follows the conservative RICE (Rest, Ice, Compression and Elevation) principle, there is no medical treatment available after the acute phase except anti-inflammatory medications and rehabilitation. The poor healing process in damaged muscle, prolonged muscle disuse, and the lack of therapeutic strategies affect the performance of daily activities and predispose to further risk of muscle injury and bone loss. Therefore, there is an unmet need to develop therapeutic strategies that can help the recovery of damaged muscle and nerve, and limit bone loss. Recently we demonstrated the potential repurposing effect of 4-aminopyridine (4-AP), a potassium channel blocker, on neurogenic muscle atrophy and functional recovery after PNI in mice. However, it is unknown whether these beneficial effects of 4-AP are present in traumatic muscle injury and muscle injury-induced bone loss. This study was thus designed to evaluate the potential therapeutic effect of 4-AP in traumatic muscle healing and PNI-induced bone loss. Standardized crush injury was performed on right sciatic nerve, biceps femoris (BF) muscle (innervated by sciatic nerve), and quadriceps femoris (QF) muscle (innervated by femoral nerve) of adult male mice (6 animals/group). Post-surgical animals were randomly assigned to normal saline and 4-AP. 4-AP (40 µg, intraperitoneal) was given daily for 21 days, and the functional recovery after injury was assessed by sciatic function index (SFI), sensation by von Frey test (VFT), and grip strength test. After 21 days, the muscles at the injury/peri-injury site were processed for histomorphometry and tibial bone density was analyzed using DEXA scanning. We found that 4-AP significantly enhanced SFI, VFT, and hind limb paw grip strength from post-injury day 7, improved BF muscle morphology, cross-sectional area, and minimum ferret diameter, and totally reversed trauma-induced muscle fiber type composition changes (type-1 vs. type-2). 4-AP-induced muscle effects were also associated with a significantly increased number of regenerating stem cells (Pax7+) and proliferating cells (Ki67+) compared with saline group. Of note, 4-AP-induced muscle effects were either less pronounced or nonsignificant in QF muscle. Most importantly, 4-AP treatment significantly reduced PNI-induced bone loss by enhancing bone mineral density and bone mineral content. In conclusion, these interesting findings, for the first time, demonstrate the potential therapeutic effect of 4-AP on the recovery of traumatic muscle and bone loss after mixed muscle and nerve injury.
• Govindappa, P. K. (2020, April). Exosome-associated ELAVL1 adversely affects cardiac macrophage-fibroblast crosstalk in diabetes. Experimental Biology.
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  BackgroundMacrophage dysfunction, inflammation and excessive fibroblast activation has been implicated in the pathogenesis of diabetes-associated heart disease. However, the paracrine mechanisms are not completely understood. We sought to unravel the influence of diabetes on exosome (Exo) transfer of Human antigen R (HuR or ELAVL1, an mRNA stabilizing protein) and its implications on cardiac fibrosis and dysfunction.Methods and ResultsWe evaluated the invitro and invivo effects of macrophage cellular- and exosome-HuR or its deficiency on paracrine mechanisms of diabetic milieu-induced inflammatory and fibrosis-related signaling. Human failing hearts show higher HuR levels as compared to non-failing hearts. Diabetic milieu (db/db mice) show increase in HuR expression in cardiac- and cultured bone marrow derived-macrophages (BMMac). High glucose (HG, 30mM) stimulates HuR nuclear-to-cytoplasmic translocation and transfer of HuR mRNA and protein into exosomes. PKC-delta inhibition blocks HG-induced HuR translocation. Exosome from macrophages exposed to diabetic milieu (HG or db/db mice) significantly increases proinflammatory and profibrogenic responses in fibroblast (invitro) or cardiac fibrosis in mice. Exo-HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with HuR-deficient-BMMac decreases ANG II-induced cardiac fibrosis and improves LV dysfunction as compared to BMMacWT administered mice.ConclusionsTo the best of our knowledge, this is the first study to demonstrate that diabetes-mediated increase in BMMac-HuR expression, its cytoplasmic translocation and exosome transfer activates fibrogenesis signaling invitro and invivo, and HuR deficiency attenuates these deleterious effects. The data suggests that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes mellitus.

Poster Presentations

• Govindappa, P. K. (2023, August). Erythropoietin level declines after acute gut injury and possible biomarkers exist to assess improvement in postoperative ileus. American Neurogastroenterology and Motility Society (ANMS). Austin.
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  Background: Intestinal motility disorders (IMDs) are poorly understood and medically complex problems affecting an increasing number of patients. Postoperative ileus (POI) is a form of IMD occurring after abdominal surgery. Erythropoietin (EPO) is a multifunctional tissue-protective cytokine that promotes recovery of the intestine from various injuries including postoperative ileus. While EPO level is known to decline in chronic inflammatory states, the level of EPO after acute gut injury and its relation to gut healing has not been studied. This study was designed to explore EPO level after POI and the effect of EPO treatment on the inflammatory cascade in a mouse model of POI.Methods: We collected serum EPO samples at baseline in wild type mice. We then performed laparotomies and induced POI in two groups: the first group was treated with EPO after POI induction and second group was treated with saline. We then collected serum and intestinal samples 24 hours after the initial injury. The tissue samples were homogenized, and serum samples were prepared for Luminex multiplex analyses of levels of EPO and other inflammatory proteins.Results: Our preliminary data shows that serum EPO level declines in POI when measured 24 hours after injury. Additionally, POI significantly upregulate inflammatory proteins c-reactive protein (CRP) and chemokine ligand 12 (CCL12) in both serum and tissue levels with significant decline of those levels after EPO treatment.Conclusions: This is the first pre-clinical study to demonstrate a novel finding of low EPO serum level after acute gut injury and how the EPO supplementation does alter the inflammatory cascade to accelerate healing as fast as 24 hours after the initial injury. Our findings suggest a novel indication for the use of EPO in POI and a marker usable to monitor recovery.
• Govindappa, P. K. (2022, May). 4-Aminopyridine promotes cutaneous reinnervation and accelerates wound healing. Experimental Biology.
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  Skin wound healing is a tightly orchestrated process between epithelial, dermal, neuronal and vascular cells, all working in concert to restore tissue integrity and skin barrier function. Here, we report the therapeutic regenerative effects of FDA-approved 4-aminopyridine (4-AP) to promote skin wound healing by accelerating re-epithelialization, wound induced hair neogenesis (WIHN), dermal collagen deposition, angiogenesis, and reinnervation. We demonstrate that 4-AP enhanced wound closure by promoting keratinocyte proliferation and migration as well as collagen deposition through myofibroblast differentiation. 4-AP treatment increased both epidermal thickness, as well as the number of hair follicles and blood vessels in healed wounds, suggesting better restoration of skin function and architecture compared to simple wound scar formation. Moreover, 4-AP enhanced reinnervation of healed wounds by promoting Schwann cell (SC) de-differentiation and secretion of neuromediators (NGF, SP) associated with regeneration. In-vitro studies using human cells demonstrated that 4-AP enhanced proliferation and migration of keratinocytes, and SCs and that 4-AP enhances cellular interactions between neuronal and non-neuronal cells to further accelerate wound healing. 4-AP induced secretion of NGF from both SCs and keratinocytes. 4-AP enhances many of the key attributes of successful wound healing and is a promising therapeutic adjuvant for skin wound healing and tissue regeneration.
• Govindappa, P. K. (2021, May). Antibiotics impair and probiotics improve functional recovery after traumatic peripheral nerve crush injury in mice. Experimental Biology.
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  Antibiotics (ABX) are widely used for the treatment of life-threatening infections and also for routine surgical prophylaxis. Compelling evidence suggests that ABX-induced alterations of gut microbiota composition and metabolism, termed dysbiosis, are linked with various disease states including gastrointestinal, metabolic, autoimmune, inflammatory, neurological, and neurodegenerative conditions. To combat the consequences of dysbiosis, nutritional interventions consisting of prebiotics and probiotics (PBX) are widely used. ABX-induced dysbiosis has recently been reported to impair neurological function after traumatic spinal cord injury. Traumatic peripheral nerve injury (TPNI) is prevalent and can result in profound neurologic impairment, prolonged recovery time, delayed return to work, and permanent disability. It is unknown whether ABX treatment-induced dysbiosis has any impact on TPNI-induced functional recovery, and if so, what role medical-grade PBX could have on TPNI recovery. In this study, ABX-induced dysbiosis (loss-of-function) and PBX-induced microbiota enrichment (gain-of-function) models were used to explore the potential role of the gut microbiome in the functional recovery of TPNI. An established mouse model of severe sciatic nerve crush injury was used for post-injury functional recovery with or without ABX, PBX, and ABX plus PBX (ABX-PBX) treatments, and stool samples were analyzed for 16S ribosomal RNA (rRNA) gene sequencing and gut microbiota compositions. Our findings demonstrate that pre-injury ABX significantly impaired post-injury functional recovery compared with the vehicle group. Conversely, pre-injury PBX significantly improved TPNI-induced functional recovery. Importantly, post-injury PBX protected against pre-injury ABX-induced functional impairment. 16S rRNA gene sequencing confirmed an ABX-induced dysbiosis and revealed that ABX-induced changes could be partially restored by PBX administration. These interesting findings demonstrate that ABX-induced gut dysbiosis exacerbates post-TPNI functional impairment and that this impairment can be prevented by early PBX administration. Since the gut microbiota are modifiable and no therapeutic treatment exists to improve patient outcomes and prevent long term disability after TPNI, clinically approved commercially available probiotics could play a significant role in the management of TPNIs, especially for those patients receiving ABX.